Gel Electrophoresis Analysis Of DNA Research Articles

The response of newly established cell lines of Spodoptera littoralis to group I and group II baculoviruses.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera littoralis multiple nucleopolyhedrovirus (SpliMNPV) belong to group I and group II nucleopolyhedroviruses, respectively and can replicate in a wide range of insect species. In this study, the ability of newly established S. littoralis cell lines to support replication of AcMNPV and SpliMNPV was examined. The microscopic observations showed that the S. littoralis cells infected with AcMNPV exhibited morphological changes such as cells breaking into small bodies and forming apoptosis-like bodies post-infection. Nuclear DNA fragmentation was observed in all AcMNPV-infected cell lines through DNA gel electrophoresis analysis. Therefore, the virus replication was unsuccessful in most of cells, which were able to abort the virus replication. On the other hand, cells that were infected with SpliMNPV did not show similar morphological changes and no small bodies were formed. In addition, SpliMNPV succeeded to infect the cells, replicate, and form viral occlusion bodies inside the infected cells. In suspension culture, S. littoralis cells, which were infected with AcMNPV, accumulated as composed balls in shaker flasks after infection overnight, with cell density decreasing dramatically. In contrast, there was no cell clumping seen in the infected cells with SpliMNPV and the uninfected cells. In conclusion, the newly established embryonic S. littoralis cells were highly susceptible to SpliMNPV, whereas the cells were non-permissive to AcMNPV, yet they still underwent programmed cell death.

Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52) is essential for preimplantation embryo development.

ABSTRACTUbiquitin A-52 residue ribosomal protein fusion product 1 (Uba52), a ubiquitin-ribosomal fusion gene, is a major source of ubiquitin protein for covalent modification of proteinaceous substrates recycled by ubiquitin-proteasome system (UPS). Its role in early embryo development has not been studied. Using the CRISPR/Cas9 gene editing tool, the objective of this study was to determine if UBA52 protein is required for mammalian embryogenesis. Matured metaphase II porcine oocytes were injected with CRISPR Cas9+guide RNAs (Uba52 gRNA) or Cas9 without gRNAs as control, followed by in vitro fertilization (IVF) and embryo culture to day 7. Injection of Cas9+gRNAs affected embryo development. On day 4 of embryo culture, the proportion of 2-, 4- and 8-cell stage embryos was significantly different between the Uba52 gRNA and control group (P<0.05), with more 8-cell stage embryos in the control and more 4- and 2-cell stage embryos in the Uba52g RNA group. This delay in the development of Uba52 gRNA embryos occurred at the transition from the 4- to 8-cell stages, around the time of major zygotic genomic activation. The percentage of blastocyst formation on day 7 and the cell number per blastocyst were significantly lower in the Uba52 gRNA group than in the control (P<0.05). Genotyping by PCR and DNA gel electrophoresis analysis showed that 91.8% of embryos that failed to develop to blastocyst had either a monoallelic or a biallelic modification of the Uba52 gene. In comparison, only 24.4% of embryos that reached blastocyst had a monoallelic modification and biallelic editing was not found in any of the blastocysts. Based on immuno-labeling intensity, both UBA52 and proteasome protein levels on days 4 and 7 of culture were significantly lower in the Uba52 gRNA group than in the control (P<0.05), in agreement with UBA52 western blotting-densitometry of day 4 embryos. Morphological examination of blastomere nuclei revealed abnormal nuclear structure in the Uba52 gRNA group, such as reduced size, irregular shapes, nucleus fragmentation and uneven DNA distribution at all stages of embryo development. Nuclear morphology studies of embryos injected with Cas9+gRNAs and co-injected with plasmid DNA encoding nuclear localized GFP further supported these observations. In conclusion, our data indicate that the Uba52 gene is essential in early embryogenesis.

Biosynthesis, characterization of magnetic iron oxide nanoparticles and evaluations of the cytotoxicity and DNA damage of human breast carcinoma cell lines

Magnetic iron oxide nanoparticles (MNPs) were synthesized using Albizia adianthifolia leaf extract as reducing and protecting agent. Colour changing, UV–Vis spectrum, X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM) confirmed the biosynthesis and characterization of MNPs. The XRD pattern revealed that MNPs are crystalline in nature. FT-IR spectral analysis showed that MNPs was capped with plant constituents. From SEM analysis, the MNPs were generally found to be spherical in shape and the size was ranged 32–100 nm. Free radical scavenging potentials of the MNPs against DPPH were confirmed based on its stable anti-oxidant effects. The synthesized MNPs were used to capture Staphylococcus aureus under the magnetic field effect. Further, it was observed that the MNPs are able to exert cytotoxic effect towards human breast (AMJ-13) and (MCF-7) cancer cells. The anti-proliferative effect of this treatment is due to cell death and inducing apoptosis. Mitochondrial membrane potential, acridine orange-propidium iodide staining assays as well as single cell and DNA gel electrophoresis analyses indicated that MNPs induce cell death only by apoptosis. The findings of present study suggest that the MNPs might be used for medicinal applications particularly for cancer therapeutics.

Potential ecological effects of Piriformospora indica, a possible biocontrol agent, in UK agricultural systems

Piriformospora indica (syn. Serendipita indica), a root endophytic fungus, was originally isolated from an arid sub-tropical soil. P. indica forms mutualistic symbioses with a broad range of host plants, increases biomass production, resistance and tolerance to fungal pathogens and abiotic stresses. These characteristics make it a very attractive component of more sustainable agriculture. So, it is desirable to understand its wider ecosystem effects. We determined how long P. indica could survive in the soil and how it interacts with other soil microorganisms and some important arable weeds.Survival of P. indica in the soil, under winter and summer conditions in the UK was tested by isolating DNA and RNA of P. indica from pots of soil which had been left open to winter-summer weather conditions without host plants, followed by PCR and reverse transcription-PCR (RT-PCR) with P. indica-specific primers. P. indica effects on other soil and root microorganisms were tested by PCR-denaturing gradient gel electrophoresis analysis of DNA extracted from soil and roots from pots in which P. indica-infected wheat had been grown. The effect of P. indica on growth of black-grass (Alopecurus myosuroides), wild-oat (Avena fatua) and cleavers (Galium aparine) was tested alone and in competition with wheat.In soil P. indica-mRNA and DNA could still be detected after eight months, but not after 15months. Soils from P. indica-inoculated pots had distinct fungal and bacterial species communities which were more diverse than non-inoculated controls. P. indica infected A. myosuroides and A. fatua but was not detected in G. aparine. The average above-ground competitiveness of the weeds with wheat was decreased.If applied to field crops in the UK, P. indica would be persistent for up to 15months and likely to alter competitive relations within vegetation. Increased soil microbial diversity during the first eight weeks after inoculation, although usually desirable, could alter soil composition or functioning.

Distribution of &lt;i&gt;Clostridium perfringens&lt;/i&gt; Isolates from Piglets in South Korea

ABSTRACTClostridium perfringens causes various digestive system disease symptoms in pigs. In the present study, 11 C. perfringens isolates were obtained from diarrheic piglets and 18 from healthy piglets. All of the C. perfringens isolates were shown to be type A using a multiplex PCR assay. The β2 toxin gene was detected in 27/29 C. perfringens isolates, i.e., 81% (9/11) of diarrheic piglets and 100% (18/18) of healthy piglets, and all of the genes had the same sequence. In conclusion, the β2 toxin gene of C. perfringens was distributed widely in Korean piglets regardless of the incidence of diarrhea, and there was no clear relationship with enteric disease. A pulsed-field gel electrophoresis analysis of DNA digested using SmaI demonstrated the non-clonal spread of C. perfringens isolates from piglets.

Antimicrobial Packaging To Retard the Growth of Spoilage Bacteria and To Reduce the Release of Volatile Metabolites in Meat Stored under Vacuum at 1°C

A nisin-EDTA solution was used for activation of the internal surface of plastic bags that were used to store beef chops at 1°C after vacuum packaging. The aim of the work was to evaluate the effect of the antimicrobial packaging on beef during storage. Volatile compounds and microbial populations were monitored after 0, 9, 20, 36, and 46 days of storage. The active packaging retarded the growth of lactic acid bacteria. Brochothrix thermosphacta was unable to grow for the whole storage time in treated samples, while the levels of Carnobacterium spp. in treated samples were below the detection limit for the first 9 days and reached loads below 5 Log CFU/cm(2) after 46 days. On the other hand, Enterobacteriaceae and Pseudomonas spp. were not affected by the use of the antimicrobial packaging and grew in all of the samples, with final populations of about 4 Log CFU/cm(2). Carnobacterium divergens was identified by PCR-denaturing gradient gel electrophoresis analysis of DNA extracted from beef after 36 days of storage. During beef storage, alcohols, aldehydes, ketones, and carboxylic acids were detected in the headspace of beef samples by solid-phase microextraction-gas chromatography-mass spectrometry analysis. The microbial metabolic activity was affected by the use of the antimicrobial film from the beginning up to 36 days with a maximum in the differences of volatile metabolites in samples analyzed at 20 days. The volatiles were also determined by electronic nose, allowing differentiation based on the time of storage and not on the type of packaging. The active packaging reduces the loads of spoilage microbial populations and the release of metabolites in the headspace of beef with a probable positive impact on meat quality.

Tunable and Regenerative DNA Zipper Based Spring

We report a DNA zipper based actuator device termed ‘DNA- spring’ with tunable and repeated cycles of extension and contraction ability. DNA zipper is a double-stranded DNA system engineered to open upon its specific interaction with appropriately designed single strand DNA (ssDNA), opening of the zipper is driven by binding energy differences between the DNA strands. The zipper system is incorporated with defined modifications to function like a spring, capable of delivering ∼9 pN force over a distance of ∼13 nm, producing ∼116 kJ/mol of work. The spring described here consists of four DNA oligonucleotides totaling 244 bases that utilize ssDNA to trigger its extension and contraction. Time-lapse fluorescence and fluorescent DNA gel electrophoresis analysis is utilized to evaluate and confirm the spring action. A second zipper incorporated into the spring provides the ability to couple/decouple to an object/substrate. Such devices would have wide application, including for conditionally triggered molecular delivery systems and as actuators in nano-devices.

Cultivation of Hard-To-Culture Subsurface Mercury-Resistant Bacteria and Discovery of New merA Gene Sequences

Mercury-resistant bacteria may be important players in mercury biogeochemistry. To assess the potential for mercury reduction by two subsurface microbial communities, resistant subpopulations and their merA genes were characterized by a combined molecular and cultivation-dependent approach. The cultivation method simulated natural conditions by using polycarbonate membranes as a growth support and a nonsterile soil slurry as a culture medium. Resistant bacteria were pregrown to microcolony-forming units (mCFU) before being plated on standard medium. Compared to direct plating, culturability was increased up to 2,800 times and numbers of mCFU were similar to the total number of mercury-resistant bacteria in the soils. Denaturing gradient gel electrophoresis analysis of DNA extracted from membranes suggested stimulation of growth of hard-to-culture bacteria during the preincubation. A total of 25 different 16S rRNA gene sequences were observed, including Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. The diversity of isolates obtained by direct plating included eight different 16S rRNA gene sequences (Alpha- and Betaproteobacteria and Actinobacteria). Partial sequencing of merA of selected isolates led to the discovery of new merA sequences. With phylum-specific merA primers, PCR products were obtained for Alpha- and Betaproteobacteria and Actinobacteria but not for Bacteroidetes and Firmicutes. The similarity to known sequences ranged between 89 and 95%. One of the sequences did not result in a match in the BLAST search. The results illustrate the power of integrating advanced cultivation methodology with molecular techniques for the characterization of the diversity of mercury-resistant populations and assessing the potential for mercury reduction in contaminated environments.

A20 overexpression under control of mouse osteocalcin promoter in MC3T3-E1 cells inhibited tumor necrosis factor-alpha-induced apoptosis

To construct an A20 expression vector under the control of mouse osteocalcin promoter (OC-A20), and investigate osteoblastic MC3T3-E1 cell line, which stably overexpresses A20 protein prevented tumor necrosis factor (TNF)-alpha-induced apoptosis. OC-A20 vector was constructed by fusing a fragment of the mouse osteocalcin gene-2 promoter with human A20 complementary DNA. Then the mouse MC3T3-E1 cell line, stably transfected by A20, was established. The expression of A20 mRNA and A20 protein in the cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. To determine the specificity of A20 expression in osteoblast, the mouse osteoblastic MC3T3-E1 cell line and mouse embryo fibroblast NIH3T3 cell line were transiently transfected with OC-A20. The anti-apoptotic role of A20 in MC3T3-E1 cells was determined by Flow cytometric analysis (FACS), terminal dUTP nick endo-labeling (TUNEL) and DNA gel electrophoresis analysis (DNA Ladder), respectively. Weak A20 expression was found in MC3T3-E1 cells with the primers of mouse A20. A20 mRNA and A20 protein expression were identified in MC3T3-E1 cells transfected with OC-A20 using RT-PCR and Western blot analysis. Only A20 mRNA expression was found in MC3T3-E1 cell after MC3T3-E1 cells and NIH3T3 cells were transient transfected with OC-A20. A decrease obviously occurred in the rate of apoptosis in the OC-A20 group compared with the empty vector (pcDNA3) group by FACS (P< 0.001). A significant increase in TUNEL positive staining was found in the pcDNA group compared with OC-A20 group (P< 0.001). Simultaneously, similar effects were demonstrated in DNA gel electrophoresis analysis. We constructed an osteoblast-specific expression vector that expressed A20 protein in MC3T3-E1 cells and confirmed that A20 protects osteoblast against TNF-alpha-induced apoptosis.

Reptile-associated Salmonellosis in Man, Italy

To the Editor: Reptiles are reservoirs of a wide variety of Salmonella serotypes, including all Salmonella enterica subspecies and S. bongori. In reptiles born in captivity or kept as pets, S. enterica subsp. enterica is frequently isolated (1). Salmonella strains are well adapted to reptiles, and they usually cause asymptomatic infections in such animals, while retaining pathogenicity for warm-blooded animals. For several years, reptiles have been recognized as a source of human salmonellosis. In North America, reptile-associated salmonellosis (RAS) has been reported, particularly in children, the elderly, or immunocompromised persons; severe and fatal infections are described occasionally (2). In contrast, only a limited amount of information on RAS is available in Europe. We report a case of RAS that occurred in an adult man in Italy. A 32-year-old man had symptoms of enteritis. For 2 weeks, he had experienced intermittent watery diarrhea, mild fever, and abdominal pain. He was then treated with ciprofloxacin, and after 15 days of treatment, he recovered from enteritis. A stool sample, collected before treatment, underwent bacteriologic analysis, and Salmonella spp. were identified biochemically (api 20E, bioMerieux, Marcy l'Etoile, France) and by a polymerase chain reaction assay specific for the invA gene of Salmonella spp (3). Since the man was a reptile owner, RAS, rather than a foodborne infection, was initially suspected. He owned several cold-blooded animals; all had been tested for Salmonella spp. (at least 3 times at 2- to 3-week intervals), and results were negative. Three weeks before the onset of enteric symptoms, he acquired a boa (Boa imperator) that was subjected to routine analysis for Salmonella spp. in our laboratories (1). Salmonella spp. were isolated from a cloacal swab of the snake. Subsequently, both the human and reptile Salmonella isolates were characterized as S. enterica serovar Paratyphi B. In addition, both strains were found to be d-tartrate–fermenting (dT+) biovars (4), susceptible to ampicillin, amoxicillin-clavulanic acid, cephalothin, ceftazidime, gentamicin, streptomycin, chloramphenicol, tetracycline, neomycin, nalidixic acid, norfloxacin, and ciprofloxacin and resistant to sulfamethoxazole and co-trimoxazole. By pulsed-field gel electrophoresis analysis of DNA, the strains displayed the same pattern, which suggests a clonal origin (4). The isolates were also assayed for virulence-associated genes. The SopE1 gene was detected in both isolates, and the avrA gene was not detected, which is consistent with an invasive pathovar of S. Paratyphi B (4). Conversely, the spvC, pef, and sef genes were not detected (5). In recent years, a general increase in RAS detection has been observed, which may be the result of the increasing diffusion of reptiles as pets and a better awareness of RAS risk. In the United States, annual reports of RAS cases are published by the Centers for Diseases Control and Prevention (2). In Europe, studies on free-living and captive reptiles have shown a high prevalence of Salmonella spp (1). Nevertheless, national surveillance systems for RAS do not exist, and epidemiologic data are incomplete. Notably, since Sweden became a member of the European Union in 1995, and the import restriction rules for reptiles were removed, a marked increase in RAS was observed in that country (6). As the deregulation of the trade in reptiles is applied, in agreement with the European Union laws, a similar scenario may be projected in other European countries. As is the case for nontyphoid salmonellosis, RAS may be underestimated, especially if patients are not hospitalized. Although a few cases of RAS have been previously reported in children in Italy (7,8), this report provides the first description of RAS in adults. S. Paratyphi B dT+, also known as S. enterica serovar Java, has been isolated in reptiles and tropical fish and has been associated with epidemics of human salmonellosis acquired from food, such as goat milk or chicken (9). The evidence shows that salmonellosis by S. Paratyphi B dT+ apparently occurs more frequently in adults (10), while so-called exotic reptile strains seem to be more prone to causing salmonellosis in children (7,8), which has led to the proposition that S. Paratyphi B dT+ strains may be highly pathogenic. By screening virulence-associated genes, both our isolates were found to be SopE1+ and avrA–, a pattern usually observed in the systemic pathovars of S. Paratyphi B (4) and associated with invasiveness, which suggests a high pathogenic potential. Accordingly, strict preventive sanitation measures should be adopted when handling reptiles (2), and reptiles should be always regarded as a potential source of pathogenic Salmonella strains for humans.

Anti-leukemia activities of Lup-28-al-20(29)-en-3-one, a lupane triterpene

The cytotoxicities of 11 lupane series triterpenes against 3 human leukemias, 2 melanomas, 2 neuroblastomas and normal fibroblast cells were examined. Lupane triterpenes with a carbonyl group at C-17 ( 7– 11) showed inhibitory effects on leukemia, melanoma and neuroblastoma cell growth. Lup-28-al-20(29)-en-3-one ( 8) markedly inhibited the cell growth of 3 leukemias to a greater extent than the other human cancers and normal lung fibroblast cells. The cytotoxicity profiles of 8 against human cancer cells showed that its cytotoxic effect against 3 lung cancer cell lines was strong and the cytotoxic effects against osteosarcoma, breast cancer and urinary bladder cancer cells were very weak. The morphological observations of leukemia nuclei and the gel electrophoresis analysis of DNA extracted from 8-treated leukemia cells revealed that 8 induced leukemia cell apoptosis. Furthermore, we investigated the cytotoxic effects of 8 on adriamycin (ADM)- and vincristine (VCR)-resistant K562 (K562/ADM and K562/VCR) cells. K562/ADM and K562/VCR cells showed greater resistance toward ADM and VCR when compared to parent K562 cells. However, 8 inhibited the drug-resistant K562 cell growth to the same extent as K562 cells by the induction of apoptosis.

4-Fluoro- N-butylphenylacetamide: a synthetic phenylacetate derivative that upregulates Bcl-X S, activates caspase cascade and induces apoptosis in human squamous lung cancer CH27 cells

Phenylacetate (PA) and related aromatic fatty acids induce antiproliferation and differentiation of cancer cell; they have potent anti-tumor properties with relatively low toxicity. To search for more potent analogues of PA, PA derivatives have been synthesized. In this study, we investigated the effects of six synthetic PA derivatives on the growth of human lung cancer cells. Results showed that the anti-proliferative effects of these synthetic compounds were strong than those of PA, 4-fluoro- N-butylphenylacetamide (H6) is the most potent compound. 4,6-Diamidino-2-phenylindole (DAPI) staining, in situ TUNEL assay and DNA gel electrophoresis analysis indicated that a marked reduction in the number of CH27 cells with H6 was related to the induction of apoptosis. The apoptosis triggered by H6 was accompanied by up-regulation of Bcl-X S, accumulation of cytosolic cytochrome c and activation of caspase cascade (caspase-9 and -3). Furthermore, H6 induces proteolytic cleavage of poly (ADP-ribose) polymerase, which followed the appearance of caspase activity and preceded DNA fragmentation. Pretreatment with caspase inhibitors markedly inhibited H6-induced caspase activity and apoptosis. These results suggest that H6 may induce apoptosis through a Bcl-X S and caspase-dependent mechanism.

Green tea polyphenols in combination with copper(II) induce apoptosis in HeLa cells

In the present investigation we report that the natural antioxidant green tea polyphenols (GTPs) could induce apoptosis in HeLa cells in the presence of copper cation. After treatment with low concentrations of GTP (5-15 μM) plus 2 μM Cu2+, HeLa cells died gradually as measured by the MTT assay. Agarose gel electrophoresis analysis of DNA extracted from HeLa cells exposed to GTP/Cu2+ showed typical internucleosomal DNA fragmentation (DNA ladder), which was also confirmed by DNA content analysis by flow cytometry. Morphological observation of HeLa cells treated with GTP/Cu2+ by transmission electronic microscopy also showed typical apoptotic features including cell shrinkage, chromatin condensation, and the formation of apoptotic bodies. Laser confocal microscopy determination of intracellular calcium level revealed that exposure to GTP/Cu2+ triggered increase of cytosolic calcium ion, which may be involved in the process of apoptosis. This evidence suggested that GTPs might be used as potential therapeutic agent for the treatment of cancer.

Comparison of different methods for the isolation and purification of total community DNA from soil

The efficiency and reproducibility of DNA extraction from soil was tested for variations in lytic and purification treatments and their effect on yield and purity of DNA. The extraction yield was improved by increasing the concentration of EDTA or monovalent ions in isolation buffers, by the introduction of mechanical lysis treatments, and by the use of ethanol precipitation in place of PEG precipitation. Purity was improved using buffers with decreasing concentration of EDTA or by reducing the ionic strength of the buffer, and by all mechanical treatments. No lytic treatment was efficient on its own, the highest purity was achieved using Crombach buffer and a combination of bead-beating with lysozyme and SDS lysis followed by potassium acetate and PEG precipitation, phenol/chloroform purification, isopropanol precipitation, and spermine–HCl precipitation. Sonication sheared the DNA more than bead-beating. Lysozyme and SDS lysis without any mechanical treatments allowed isolation of larger fragments (40–90 kb). Denaturing gradient gel electrophoresis analysis of DNA isolated using a range of lytic treatments revealed alterations in band patterns which might reflect differences in the efficiency of lytic treatments.

The Intrinsic Curvature of a 51 bp K-DNA Fragment of Leishmania tarentolae: A Molecular Model

DNA intrinsic structure and curvature is a subject of debate because of the importance of these attributes in processes such as DNA packaging, transcription, and gene regulation. X- ray crystallography of DNA single crystals has provided a wealth of information about the local, short range conformational features of DNA. On the other hand, gel electrophoresis analysis of DNA has not only uncovered the macroscopic curvature of DNA but it also provides most of the available data on DNA intrinsic curvature. However, gel electrophoresis can not identify features of DNA structure at the nucleotide or atomic level. In order to address the problem of DNA intrinsic curvature in an attempt to bridge the gap between X- ray crystallography and gel electrophoresis, we use the computational method of molecular dynamics (MD). In this study, we report the results of 2.0 ns MD simulations on a 51 bp fragment of the K-DNA of Leishmania tarentolae containing several A-tracts. The K-DNA double helix is very stable and remains in an intermediate state between the canonical A and B forms of the duplex. The magnitude of global curvature (75°) agrees well with the experimental estimate (72°) available. Analysis of local (every base triplet) and sublocal (every helix tum) curvature shows that the 51 bp K-DNA fragment has curvature features also present in the Wedge, Junction and Calladine's models of DNA intrinsic curvature. We further characterize the flexibility of individual nucleotides in the molecule and find the sugar flexibility within the A-tracts to be strongly correlated with the pattern of A-tract cleavage by the hydroxyl radical. Differential curvature and flexibility at the 5′ and 3′ junctions between A- tracts and general-sequence DNA are found to modulate the global curvature of the K-DNA fragment.